A full bibliographic citation of the references cited in this application can be found in the section preceding the claims.
The present invention generally relates to markets where the detection or purification of molecules is involved. Representative markets include in vitro and in vivo diagnostics, research products, clinical products, clinical research products, pharmaceuticals and many industrial markets. These markets necessarily require a tight binding and specific affinity ligand that recognizes the biomolecule of interest. Given the importance of affinity recognition for proteins in a wide variety of markets, new methods for generating xe2x80x9cimmunoaffinityxe2x80x9d or xe2x80x9cmacromolecular recognitionxe2x80x9d ligands using DNA technology are sought.
The production of proteins and peptides by recombinant DNA technology is now relatively common. Recombinant expression of peptides often is accomplished by inserting a DNA sequence encoding the desired peptide into an expression vector. The expression vector generally contains regulatory sequences which are recognized by the host cell, and which provide for transcription and translation of the inserted DNA to produce the peptide. The expression vector is inserted into a suitable whole cell, typically a procaryotic organism, in culture. The expression vector also usually includes a selectable marker, so that one may identify those cells which have been transformed successfully and carry the vector, and separate them from those which do not carry the vector.
Peptides may be expressed either directly or in the form of a fusion protein. Direct expression involves the production of the desired protein or peptide without modification. However, this form of expression often results in low yields and degradation of the product, particularly with small peptides.
One method commonly employed to increase the yield of the desired peptide is to express it as part of a fusion protein. In this approach, a leader protein, hereafter referred to as a carrier segment, is selected which is expressed easily in the host of choice. Often this protein is native to the host, as in the case of xcex2-galactosidase. An expression vector encoding the carrier segment then is modified using standard molecular cloning techniques to express the desired peptide linked to the carrier segment. Most often, the peptide is linked to the carboxy terminus of the carrier segment but, in principle, it could be linked anywhere through a normal peptide linkage.
The expression of the peptide as a fusion protein with the carrier segment may imbue it with new, favorable properties. For instance, the fusion protein can be injected into a host animal to create antibodies to the peptide or to produce a vaccine. The technology of using fusion proteins as antigens is well-known to the art (Current Protocols, 1994, Chapter 16). Other favorable properties of the fusion protein may include ease of purification, the ability to be immobilized on surfaces, and the creation of bifunctional molecules (Santo, C. et al. 1992).
Although peptides with desired additional properties are commonly produced by the use of fusion proteins, it is also common to produce a peptide separately through chemical synthesis. The peptide then can be covalently coupled to purified carrier segments using chemical crosslinking agents. The resultant carrier protein conjugate can be used in many of the same applications as the fusion protein described above.
For example, peptides are sometimes covalently coupled to keyhole limpet hemocyanin for immunizations or to alkaline phosphatase for use as a detection reagent. In the case of the fusion protein, the only molecules which can be linked to the carrier segment are peptides, and the linkage must be through the normal polypeptide backbone.
When the second molecule is coupled chemically to the carrier segment after expression, the nature of both the second molecules, called ligands, and of the chemical linkages to the carrier segment is much broader. Any ligand and crosslinker which is chemically allowed can be contemplated.
In some cases, covalent association of the ligand (peptide, hapten, or other) with the carrier segment is not required to be effective. The two molecules associate with each other by any number of means. In this case, the mixture of the two is called a carrier protein complex.
The following paragraphs illustrate some uses of prior art fusion proteins. For example, U.S. Pat. No. 4,743,679 to Cohen et al. discloses the expression of epidermal growth factor (EGF) as a fusion protein with a leader sequence of up to 200 amino acids (preferably up to 75). The fusion protein is expressed in bacteria as an insoluble inclusion body.
U.S. Pat. No. 5,302,526 to Keck et al. is directed to recombinant DNA encoding amphophilic leader sequences (carrier segments) for the production and purification of fusion proteins. The polypeptide comprises an amphophilic helix designed to have hydrophilic charged amino acid residues on one side and nonpolar amino acid residues on the other side of the helix. When a gene encoding a protein of interest is attached to the helix, an inclusion body is formed. The inclusion bodies may be collected and purified.
U.S. Pat. No. 5,322,930 to Tarnowski et al. describes a method for expressing proteins as fusion proteins by using the portion of human pro-atrial natriuretic peptide (proATP) as the carrier for a heterologous peptide, wherein each of the Glu residues normally present in the proATP protein portion is altered to Gln.
U.S. Pat. No. 5,008,373 to Kingsman et al. describes a fusion protein system useful in vaccines or in diagnostic or purification applications. The fusion protein includes a first amino acid sequence derived from a retrotransposon or an RNA retrovirus encoded for by a yeast TYA gene sequence. The second amino acid sequence is a biologically active amino acid sequence, acting as the antigen.
U.S. Pat. No. 5,322,769 to Bolling, et al. is directed to a method for using CKS fusion proteins in assays.
Lin et al. (1987) disclose the use as an antigen of a fusion protein containing a carrier segment consisting of the gene 10 molecule of phage T7.
A major problem with prior art carrier segments is that the carrier segments also are known to be antigenic; that is, antibodies are produced in response to the carrier segment. Thus, there is a competition between the production of antibodies to the desired ligand and the production of antibodies to the carrier segment which may result in a lower production of antibodies with specificity for the target segment. Since the immune system usually reacts to surface exposed peptide segments which are often charged, it is likely that the existence of charged residues on the carrier segment exacerbates such problems with antigenicity.
Another problem with prior art carrier segments is that they often require the use of adjuvants. Adjuvants serve a variety of purposes (Klein, 1990). Adjuvants trap the antigen by causing the formation of an emulsion, precipitate or small vesicles at the injection site from which the antigen is released slowly over a prolonged period. The clearance of the antigen is thus delayed and the organism""s exposure to the antigen is lengthened. Adjuvants also stimulate the nonspecific migration of cells to the site of antigen injection and increase the probability of interaction of the antigen with cells of the immune system. Further, adjuvants increase antigen dispersion in the recipient""s body by continually delivering the antigen in small amounts from the injection site to the regional lymph nodes or spleen. Some adjuvants also have a mitogenic effect and so stimulate the proliferation of lymphocytes nonspecifically. Some adjuvants also help to stimulate lymphocytes by activating adenylate cyclase and other chemical messengers. Adjuvants may increase the probability of contact among T- and B-cells, macrophages, and antigens through the activation of lymphocyte-trapping mechanisms. The main problem with adjuvants is that some of the most effective ones tend to be toxic to and/or cause lesions in the host organism. They also can be difficult to handle.
The present invention is directed to a fusion protein carrier segment comprising a non-naturally occurring, hydrophobic, sparingly soluble amino acid sequence.
The present invention is also directed to a substantially nonantigenic fusion protein carrier segment comprising a non-naturally occurring, hydrophobic sparingly soluble amino acid sequence having a length at least about 65 amino acids long, wherein the carrier segment comprises no more than approximately 5% of the following amino acids arginine, lysine, aspartic acid, glutamic acid, cysteine, tryptophan and methionine.
The present invention further is directed to a carrier protein conjugate comprising a first amino acid sequence, wherein the first amino acid sequence is at least about 65 amino acids long and the first amino acid sequence lacks at least two of the following amino acids selected from the group consisting of the following negatively or positively charged side chains of amino acids: arginine, lysine, aspartic acid, glutamic acid; or uncharged side chains of the following amino acids: cysteine, tryptophan and methionine (with the exception of methionine at the amino terminal site); and a ligand fused to the first amino acid sequence.
Further, the present invention is directed to an amino acid sequence as illustrated in FIG. 1 [SEQ. ID. 1] or to an amino acid sequence as illustrated in FIG. 3 [SEQ. ID. 2].
The present invention also is directed to a non-naturally occurring fusion compound comprising an amino acid sequence as illustrated in FIG. 1 [SEQ. ID. 1] linked to a ligand.
The present invention also is directed to an expression vector comprising an amino acid sequence in a single reading frame with a coding sequence for an antigen, wherein the expression vector expresses a particle-forming fusion protein encoded by the amino acid sequence and a ligand, wherein the amino acid sequence is a non-naturally occurring, hydrophobic, sparingly soluble amino acid sequence wherein the amino acid sequence lacks at least two of the following amino acids selected from the group consisting of the following negatively or positively charged side chains of amino acids: arginine, lysine, aspartic acid, glutamic acid; or uncharged side chains of the following amino acids: cysteine, tryptophan and methionine (with the exception of methionine at the amino terminal site).
The present invention further is directed to an adjuvant for administering a high molecular weight protein to a host animal, the adjuvant comprising a non-naturally occurring, hydrophobic, sparingly soluble amino acid sequence.
The present invention also is directed to a process for producing an antibody to a ligand comprising: a) fusing a carrier segment comprising a non-naturally occurring hydrophobic sparingly soluble amino acid sequence to the ligand to form a fusion protein or carrier protein conjugate or carrier protein complex; b) presenting the antigen by in vivo or in vitro means; and c) producing the antibody to the ligand by in vivo or in vitro means.
The present invention also is directed to an assay for determining the concentration of an anti-antigen antibody in a test sample, wherein (a) at least a carrier protein conjugate is attached to a solid phase as capture reagent and is contacted with the test sample for a time and under conditions suitable for antigen/antibody of complexes to occur, and (b) an indicator reagent comprised of a signal generating compound and a specific binding member for the analyte is contacted with the complexes for time sufficient for a reaction to occur, wherein the signal generated is an indication of the presence of the anti-analyte antibody in the test sample. The improvement comprises attaching a fusion protein carrier segment comprising a non-naturally occurring hydrophobic sparingly soluble amino acid sequence to the solid phase as the capture reagent.
The present invention is further directed to a process for vaccinating a host vertebrate animal with an immunogen, comprising: (a) preparing an immunogen comprising a substantially nonantigenic fusion protein carrier segment containing a non-naturally occurring, hydrophobic, sparingly soluble amino acid sequence having a length at least about 65 amino acids long wherein the amino acid sequence lacks at least two of the following amino acids selected from the group consisting of the following negatively or positively charged side chains of amino acids: arginine, lysine, aspartic acid, glutamic acid; or uncharged side chains of the following amino acids: cysteine, tryptophan and methionine (with the exception of methionine at the amino terminal site); and (b) administering the carrier protein conjugate to the host vertebrate animal.
The present invention also is directed to a process for vaccinating a host vertebrate animal with an immunogen, comprising: (a) forming an immunogen comprising a non-naturally occurring carrier protein conjugate containing an amino acid sequence as illustrated in FIG. 1 [SEQ. ID. 1] and a ligand fused to the amino acid sequence; and (b) administering the carrier protein conjugate to the host vertebrate animal.
Further, the present invention is directed to a process for immunopurifying antibodies from a pool of antibody material, comprising: (a) forming a substantially nonantigenic fusion protein carrier segment containing a non-naturally occurring, hydrophobic, sparingly soluble amino acid sequence having a length at least about 65 amino acids long wherein the amino acid sequence lacks at least two of the following amino acids selected from the group consisting of the following negatively or positively charged side chains of amino acids: arginine, lysine, aspartic acid, glutamic acid; or uncharged side chains of the following amino acids: cysteine, tryptophan and methionine (with the exception of methionine at the amino terminal site); (b) incubating the antibody material with the carrier protein conjugate under binding conditions to bind the antibody material to the carrier protein conjugate to form an antibody/conjugate complex; (c) separating out the antibody/conjugate complex; (d) dissociating the antibody from the antibody/conjugate complex; and (e) separating the antibody from the antibody/conjugate complex.
Further, the present invention is directed to a process for producing peptides, comprising: (a) incubating a fusion protein containing a fusion protein carrier segment comprising a non-naturally occurring hydrophobic sparingly soluble amino acid sequence, wherein the amino acid sequence lacks at least two of the following amino acids selected from the group consisting of the following negatively or positively charged side chains of amino acids: arginine, lysine, aspartic acid, glutamic acid; or uncharged side chains of the following amino acids: cysteine, tryptophan and methionine (with the exception of methionine at the amino terminal site), under conditions where the peptide bonds on the fusion protein are cleaved; and (b) separating out the desired peptides.
Further, the present invention is directed to a protease inhibitor comprising a fusion protein containing a fusion protein carrier segment comprising a non-naturally occurring hydrophobic sparingly soluble amino acid sequence and a cleavage site for a protease, wherein the fusion protein is isolated and used as a competitive inhibitory substrate.
Further still, the present invention is directed to a solid support having absorbed thereon a carrier protein conjugate comprising: (a) a first amino acid carrier sequence, wherein the first amino acid sequence is at least about 65 amino acids long and the amino acid sequence lacks at least two of the following amino acids selected from the group consisting of the following negatively or positively charged side chains of amino acids: arginine, lysine, aspartic acid, glutamic acid; or uncharged side chains of the following amino acids: cysteine, tryptophan and methionine (with the exception of methionine at the amino terminal site); and (b) a ligand fused to the first amino acid carrier sequence.
The present invention also is directed to an assay for determining the concentration of an enzyme in a test sample, wherein (a) at least a carrier protein conjugate containing a ligand upon which the enzyme can act is attached to a solid phase and is contacted with the test sample for a time and under conditions suitable for the enzyme to act upon the substrate, and (b) indicator reagents capable of generating a signal change in response to the enzyme-catalyzed modification of the immobilized substrate is contacted with the complexes for time sufficient for a reaction to occur, wherein the signal generated is an indication of the presence of the enzyme in the test sample. The improvement comprises attaching a recombinant fusion protein carrier segment comprising a non-naturally occurring hydrophobic sparingly soluble amino acid sequence to the solid phase as the capture reagent.
The present invention also is directed to a test kit for use in detecting the presence of anti-antigen antibodies in a test sample, which test kit contains a container containing at least one protein specific for the anti-antigen antibody and, wherein the improvement comprises a container containing a non-naturally occurring hydrophobic sparingly soluble amino acid sequence, which is specific for the anti-antigen antibody.
The present invention also is directed to an immunoassay kit for detecting a specific antigen, comprising in separate containers:
a. a solid support having bound thereto one or more antibodies produced according to the process described above that will specifically react with a desired antigen;
b. a buffer to remove unbound proteins;
c. solutions for detection of bound analyte; and
d. instructions for use.
The advantages of the present invention are specifically directed to the novel carrier segment. Some of the advantages of the carrier segment are as follows:
Low Antigenicity: One of the primary goals of the present invention is to make antibodies to a ligand. In order to have the best opportunity to make the antibodies to the ligand, the carrier segment portion of the fusion protein must be relatively nonantigenic to the animal. Typically, the immune system in a host body will target foreign materials, i.e., antigens presented to the system. When an antigen such as a fusion protein enters the body, the immune system will seek out the most antigenic moiety to attack. If the carrier segment happens to contain the most notable antigen, the immune system will preferentially target the carrier segment and have little response to the target ligand segment of choice. This is often the case primarily because the carrier segment tends to be the larger segment, usually 300 or more amino acids. In naturally occurring proteins used as carrier segments, there are also a large number of charged amino acids.
Charged amino acid sequences are more likely to be surface-exposed and antigenic than are hydrophobic amino acid sequences. Thus, the carrier segment has more potential epitopes, and consists of residues which frequently are more easily recognized as foreign or dangerous by the immune system. By making the carrier segment small and very low in antigenicity, the immune system will be more likely to attack the ligand and produce antibodies to the ligand.
For the purposes of the present invention, low antigenicity is defined as failure of the carrier segment to induce formation of greater than 50% of the specific antibody generated in response to three or more injections into chickens, mice and rabbits of a fusion protein consisting of the carrier segment fused to a ligand having the sequence: H2N-Lys Met Ala Glu Asp Asp Pro Tyr Leu Gly Arg Pro Glu Gln Met [SEQ. ID. 4]
One way of reducing the antigenicity of the carrier segment is to reduce its size. This allows the attached ligand to comprise a significantly greater fraction of the total fusion protein. Typically, the carrier segment contains between approximately 200 and 500 amino acids in length. However, by reducing the length of the segment to an amino acid length of approximately 100 or fewer amino acids and removing many charged amino acids, the carrier segment is less effectively displayed to the immune response in the host organism. The carrier segment should be at least 65 amino acids long, and preferably 100 amino acids long. At the very least, the length of the carrier peptide should be sufficiently long to allow effective expression within the procaryotic host expression organism.
Low Solubility: A major advantage of the present carrier segment is that it is sparingly or slowly soluble. This results in an advantage for rapid purification since injection quality immunogen containing low levels of pyrogen can be prepared using simple washes as opposed to conventional methods such as chromatography, thus resulting in savings in both time and equipment. Additionally, the sequestration of the peptides in inclusion bodies when expressed may protect labile sequences from proteolytic attack.
No Need for Adjuvants: An additional advantage of the carrier segment is that it may act as its own adjuvant as well as a conventional carrier protein due to various properties of the carrier segment and of conjugates containing the segment, including low solubility and mitogenic effects.
A typical way researchers currently prepare antigens for production of antibodies is to chemically synthesize the ligand or peptide using techniques known to the art, and then to chemically crosslink it to a larger protein, known as a carrier protein. The function of the carrier protein is to elicit T-cell help. T-cell help is important in producing an effective antibody response within an animal. However, many ligands and peptides are too small to both present the desired antigenic site and to include a segment which allows the material to elicit T-cell help. Certain proteins, such as keyhole limpet hemocyanin, are known to the art to contain effective sites for eliciting T-cell help, and are thus often coupled to small ligands for this purpose. Unfortunately, as discussed previously, many of these also are antigenic and can dominate the antibody response. The carrier segment described elicits the necessary T-cell help without dominating the antibody response, and thus is a superior carrier protein.
A typical way researchers currently prepare a dosage of the antigen for injection for the production of antibodies is to formulate it in any number of suspension, precipitate or emulsion forms, called adjuvants. Adjuvants can augment the immune response as the result of the combined effect of several factors. If the antigen were injected into the host animal as is, it would be in a soluble form and would be quickly dispersed from the site of injection and cleared from the body Since the immune system needs time to recruit immune cells to the site of injection and to mount a response, the quick dispersal and clearance of antigen is detrimental to the immune response. Formulation of the antigen so that it is trapped at the site of injection is an effort to keep the antigen localized long enough for the immune system to locate it and mount a response. In most cases, the antigen is not trapped long enough at the site to elicit a robust immune response, so multiple injections over a period of several weeks are required. Additionally, recruitment of immune cells to the site of injection usually requires some local nonspecific inflammation. For this reason, some adjuvant formulations also contain an irritant for this purpose. Adjuvants also may trap the recruited immune cells, thereby enhancing the interactions of the immune cells. Finally, adjuvants may stimulate proliferation of the immune cells.
The low solubility of the carrier segment and of conjugates containing it creates conditions at the injection site that are sufficient for mounting an immune response; namely, that some sort of nonspecific inflammatory response is created that recruits immune cells to the location, that antigen is sequestered at the site long enough for an immune response to be mounted, that interactions between the immune cells may be potentiated, and that immune cell proliferation is stimulated. If the solubility were too low, or if the antigen were not accessible to the immune system, no response would be achieved; thus, the solubility of the carrier segment is thought to be in an optimal range which balances the need for sequestration against the requirement for slight solubility and effective antigen display. Thus, the carrier segment has been designed to provide all functions required for eliciting an immune response; recruitment of immune cells to the injection site, sequestration of antigen at the site, eliciting T-cell help without dominating the antibody response, and effective antigen display to the immune system. The advantages of this system over prior art is that it eliminates the need for a step involving coupling to a carrier protein, and a step involving formulation in an adjuvant. Additionally, a single injection of antigen is sufficient to elicit an ample immune response.
Most adjuvants induce the formation of granulomas in animals and humans. For this reason, most adjuvants are not allowed for use in humans although they are allowed for use in animals (Klein, 1990). Therefore, a system which does not require the use of adjuvants is not only more humane, but may have significant benefits specifically in the field of vaccine development.
Using the carrier segment described, antibody isotype response may be able to be altered. Different isotypes or classes of antibody heavy chains are known to have unequal effector functions, such as the capacity to fix complement, and unequal capacity to bind available affinity resins, such as Protein A. Thus, antibodies with the same antigen specificity but of a different isotype have disparate effector functions and resin-binding affinities. The system of the present invention induces different antibody isotype profiles in response to different concentrations of the antigen. This suggests that a system may be developed which will allow the regulation of the predominant isotype or class of the antibodies which are made using this technology.
Excellent Expression: The property of excellent expression simplifies the production of a fusion protein by allowing small fermentations to be performed for the isolation of sufficient antigens for immunizations as well as subsequent uses such as validation assays and affinity purification regimens. It is logical to assume that enough of the fusion protein must be made in order for it to be injected into the host animal for antibody production. Usually, research requires the injection of more than one host animal. The carrier segment of the present invention is advantageous in that it is able to be expressed at a very high rate, e.g., xe2x89xa75 mg/l in standard culture medium, in bacteria. Another advantage is that it allows the accumulation of the peptide without rapid degradation, thus allowing intact fusion proteins to be isolated.
Works with a Variety of Target Ligands: Another advantage of the carrier segment of the present invention is that it is relatively nonspecific in terms of attaching to target ligands. Therefore, virtually any type of ligand can be fused to the nonantigenic, sparingly soluble carrier segment to produce a useful immunogen in which the carrier segment is nonantigenic and the desired peptide is selectively displayed to the immune system.
Broad Utility as an Antigen: Another advantage of the present invention is that the fusion protein created by the carrier segment of the present invention creates enhanced antigenic responses in most antibody producing species. Typical species include vertebrates such as the mouse, rat, rabbit, chicken, goat, sheep, donkey, horse and human. The fusion protein has been shown to be effective using a variety of routes of injection as discussed infra.
Yield of Specific Antibodies Can be High: Another advantage of the present invention is that the ratio of specific antibodies to the ligand of interest compared to those antibodies directed against the carrier segment of the fusion conjugate is high. Typical carriers, such as KLH or BSA, are both antigenic in nature and much larger in size than the ligand. Thus, many or most of the antibodies produced in response to immunization with such a compound are directed against the carrier segment.
The present invention constitutes a much smaller carrier segment which has low antigenicity. Since there are both fewer epitopes and less antigenic epitopes presented to the immune system with the current invention, there is a net effect of making the ligand immunodominant. This results in a more robust immune response to the ligand of interest.
Production of Peptides: The present invention also advantageously allows the rapid production of large quantities of short peptides of defined sequences to be expressed as an insoluble fusion protein. The hydrophobic carrier segment can be cleaved and removed, or left attached. In addition to the uses listed above, leaving the carrier segment attached enables the peptide to be immobilized on hydrophobic surfaces, which may have utility for a wide variety of applications. The peptides can be used in all ways other short peptides are used, such as the neutralization of antibodies, direct immunization as discussed above, conjugation to other carriers for immunization, epitope mapping, as therapeutic agents, substrates for the assays of enzymes, and other applications. As is well-known in the prior art, fusion proteins can be processed by a variety of reagents to yield the desired peptide sequence separated from the carrier segment. An advantage of this invention is that removal of the carrier segment is facilitated by the low solubility of the carrier segment when the desired peptide is cleaved off.
Other advantages of the present invention over conventional peptide synthesis include lower cost of production of small quantities of peptides of defined sequences. Further, no toxic reagents are required in the process of the present invention. There also is less of a chance of modification of amino acid side chains, which has been demonstrated to be a problem with conventionally synthesized peptides.
The present invention also offers significant improvements over the current technology for making antibodies to peptides or proteins. The present invention makes it easier for an antibody to be made from a peptide sequence of a protein without requiring knowledge of the entire sequence of the target protein, as well as making antibodies with high affinity, specificity, and avidity to both the peptide and the parent protein from which the peptide was derived. Therefore, the basic problems involved with immunization, immunoaffinity purification of antibodies from polyclonal sera, peptide production and epitope mapping or identification can all be accomplished in facile, inexpensive, safe ways using equipment and methods very familiar to biologists not accustomed to working with peptides or synthesizing peptides.
The product of the present invention is designed to 1) produce a safe vaccine against infectious diseases, and 2) synthesize defined proteins against which antibodies can be raised for experimental, industrial, pharmaceutical and diagnostic purposes.
The present invention also is useful in the production of diagnostic reagents for use in diagnostic assays. The fusion protein, comprising the combination of carrier sequence and ligand, can be dispersed or immobilized on a variety of solid supports such as test tubes, microliter plate wells, dipsticks and the like. Moreover, using this technology makes it possible to generate longer peptides than feasible under standard chemical synthetic methods.
These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention, the examples, and the attached drawings and photographs.